Treatment of hepatitis B infection with thymosin alpha 1 in combination with lamivudine or in combination with lamivudine and famciclovir

ABSTRACT

A method of treatment of hepatitis B virus (HBV) infection in a patient by administering to the patient a drug regimen including an antiviral-effective amount of thymosin alpha 1 (Tα1), an antiviral-effective amount of lamivudine, and optionally an antiviral-effective amount of famciclovir is disclosed.

This application claims benefit of Provisional No. 60/101,771 filed Sep.25, 1998.

FIELD OF THE INVENTION

The present invention relates to the field of hepatitis B treatment.

BACKGROUND OF THE INVENTION

Chronic hepatitis B virus (HBV) infection is a serious global healthproblem affecting around 300 million individuals. Among them,approximately seventy-five percent are believed to be Asian. It also isestimated that 25-40% of these HBV carriers will die of cirrhosis orhepatocellular carcinoma.

In contrast to the course of the disease in Caucasians, the naturalhistory of chronic HBV infection in Asians is characterized by aninitial active viral replicative state with minimal liver damage (immunetolerance phase), followed by an active immune clearance phase withchronic active hepatitis, and later an inactive HBV non-replicativephase with the development of cirrhosis that may be complicated byhepatocellular carcinoma (HCC). In addition, a fourth phase,characterized by viremia and chronic active hepatitis in the absence ofHBE antigenaemia may follow. The main aim of current treatment is tosuppress HBV replication before there is any significant irreversibleliver disease. As most of the liver damage occurs during the immuneclearance phase (when HBV replication is being suppressedspontaneously), it would be ideal to suppress HBV replication in anearlier phase (immune tolerant phase).

So far, therapeutic trials have mainly been directed toward utilizationof anti-viral agents, immunomodulators, immunosuppressives or certainspecific combinations of these. At present, interferon-alpha is the onlytherapeutic approach that has had regulatory approval in a number ofcountries. However, interferon therapy has been reported to producesustained clearance of hepatitis B e antigen (HBeAg) in 30-40% of NorthAmerica and European patients, but only 25-65% of these patientsultimately cleared hepatitis B surface antigen (HBsAg). The responserate in Asian patients is lower: approximately 15-20% will clear HBeAg,of these only approximately 10% will clear HBsAg. One of the factorsthat affects the antiviral effects of these immunomodulatory agents isthe high pretreatment HBV DNA level. Recently, second generationnucleoside analogs, such as lamivudine and famciclovir, have been shownto be effective in suppressing HBV replication with a good safetyprofile. However the nucleoside analogs have not been shown to maintaindurable HBV DNA suppression once therapy is removed. Hence, thecombination therapy goal is suppression and clearance.

There remains an urgent need in the art for effective anti-viral therapyagainst chronic HBV infection.

Thymosin alpha 1 (Tα1) initially isolated from Thymus Fraction 5 (TF5),has been sequenced and chemically synthesized. Tα1 is a 28 amino acidpeptide with a molecular weight of 3108, which has shown activitysimilar to TF5 in modulating the maturation of T cells. Tα1 caninfluence immunoregulatory T cell function, promote interferon-alpha,interferon-gamma, and IL2 and IL3 production by normal humanlymphocytes, and increase lymphocyte IL2 receptor expression.

There is evidence to suggest that Tα1 may influence recruitment ofpre-NK cells, which would then become cytotoxic after exposure tointerferon. Tα1 may also directly influence the lytic activity of matureNK cells. Recent investigations have shown that Tα1 enhances bothallogenic and autologous human mixed lymphocyte reactions by activationof the T4 (helper/inducer)cells. This provides additional evidence thatT4 cell may be the predominant target cell for the biological effect ofTα1.

Clinical trials of Tα1 as primary or adjunctive therapy for treatment ofHBV infection indicate that it enhances immune responsiveness andaugments specific lymphocyte parameters to significantly higher levels.

TF5 has been reported to decrease spontaneous cell-mediated lysis ofhepatocytes using Peripheral Blood Mononuclear Cells (PBM) from patientswith CHB. No effect on cytotoxicity was seen with TF5 treated controlPBM. Additional studies showed that TF5 increased Con A-inducedsuppressor function in PBM from patients with CHB. Tα1 has been shown toenhance in vivo production of anti-HBs following Heptavax-B vaccinationin previously non-reactive hemodialysis patients.

A controlled investigation was initiated in which 6 chronic woodchuckhepatitis virus (WHV)-carrier woodchucks were given twice weeklysubcutaneous injections of Tα1 (10 μg/Kg) for 28 weeks. At theconclusion of the treatment, WHV DNA levels were undetectable in 4 ofthe treated animals, and were depressed 100-fold in the remaining 2animals. Liver biopsy specimens obtained at the conclusion of treatmentrevealed a 50 to 300-fold decrease in the levels of WHV DNA replicationintermediates in the 4 animals in which serum WHV DNA wasundetectable-but only a small change from the pretreatment levels in theother 2 animals. No changes were identified in serum WHV DNA levels, orin tissue WHV DNA replication intermediates before or after treatment inany of the 6 untreated control animals.

In a US Phase II trial of Thymosin fraction 5 (TF5) and Thymosin alpha 1(Tα1) in the treatment of CHB, 12 patients received TF5 or Tα1 and 8patients received placebo twice weekly for six months. By the conclusionof the study (1 year), serum aminotransferase levels had improvedsignificantly in the Thymosin e1-treated patients, but not in theplacebo group. Nine (75%) of the Thymosin α1-treated patients and 2(25%) patients given placebo cleared serum HBV DNA (p<0.004, Fisher'sexact test). Response to Thymosin al therapy was associated withsignificant improvements in peripheral blood lymphocyte, CD3 and CD4counts, and in vitro production of interferon-gamma over initial values.No side effects were observed in patients given Tα1, and severalpatients given TF5 experienced induration and erythema at the injectionsites. About 78% of the responders had a sustained remission with normalALT levels and negative serum HBV DNA (using PCR) and HBeAg.

Subsequently a U.S. Phase III CHB multi center, placebo controlled,double-blind study was conducted in 99 patients that were serum HBV DNAand HBeAg positive. All patients were HbsAg(+) for at least 6 monthswith persistent ALT elevation. Following a 3 month screening period, 50patients were randomized to receive Tα1 (1.6 mg, S.C. 2×/week) and 49 toreceive placebo (mannitol, NaPO4; S.C. 2×/week) for 6 months andfollowed at month intervals for 6 months. Thirty eight Tα1 treatedpatients and 32 patients given placebo were followed after the 1 yrstudy for 23+7 (SD) months respectively. Two patients were removed fromthe study after randomization but prior to treatment. Results arepresented for 49 patients in the Tα1 group and 48 patients in theplacebo group. There were no statistically significant differences inALT values, HBV DNA levels and histological findings between the twogroups at inclusion. A complete response (CR) to treatment was definedas a sustained serum HBV DNA(−) status (2 negative results at least 3months apart) during the 1 year study with negative HBV DNA and HBeAg at12 months. A delayed response(DR) was determined as a sustained HBVDNA(−) status achieved after 12 months with a negative HBV DNA and HBeAgat last assessment. An incomplete response (IR) was characterized as asustained HBV DNA(−) status during the study or post study period withcontinued presence of HBeAg. The response to treatment was as follows:

Group (n) CR (%) DR (%) IR (%) Tal(49) 7(14%) 5(10%) 0(0%) Placebo(48)2(4%) 4(8%) 2(4%) p value 0.084 ns ns

A total of 12 (25%) patients treated with Tα1 and 6(13%)patients givenplacebo (p<0.11) showed a sustained loss of HBV DNA with negative HBeAgduring the 1yr study or post study follow up. Lamivudine is a reversetranscriptase inhibitor. As the activity of reverse transcriptase isnecessary to transcribe the HBV-RNA pre-genome into HBV-DNA, a targetfor selective inhibition of viral replication is present. Selection ofthe negative enantiomer of 2′3′-dideoxy-3′-thiacytidine (lamivudine)enhances the antiviral activity but limits toxicity, probably becausethe negative enantiomer has difficulty in crossing the mitochondrialdouble membrane. The antiviral activity of lamivudine has been shown inHBV transfected cell lines, the duck model and in chronically infectedchimpanzees. The drug has been used for up to 2 years in HIV-infectedpatients without significant toxicity.

Famciclovir is the oral form of penciclovir (BRL39123), a novelnucleoside analogue which has proven efficacy against the herpes simplexand zoster viruses. Famciclovir, the diacetylester of 6-deoxypenciclovir (asynthetic acyclic guanine derivative), is a prodrug ofpenciclovir (active component). Conversion to penciclovir takes placepartly in the intestinal wall, where one ester group is removed, and iscompleted in the liver, where the remaining ester group is removed andoxidation occurs at the 6-position of the purine base.

Studies in volunteers have shown that famciclovir is well absorbed andproduces penciclovir concentrations in the blood (Cmax 21.73microgram/ml after a 500 mg oral famciclovir dose) superior to thoseobtained following oral administration of penciclovir alone (Cmaxapproximately 0.14 microgram/ml after a 5 mg/kg dose). Single and repeatdoses of famciclovir have been given to human volunteers at daily dosesup to 1000 mg three times daily for six days. There was no evidence ofdrug-related adverse effects on clinical laboratory values, bloodpressure, heart rates or electrocardiograph (ECG) measurements.

An integrated safety evaluation involving 11 completed, randomized,double-blind clinical trials and 2 open trials (with more than 3000patients tested) showed that the frequency of adverse experiences andlaboratory abnormalities (hematology, clinical chemistry and urinalysisparameters) were similar in both famciclovir and placebo recipients. Themost common adverse experiences were headache, nausea and diarrhea.

Penciclovir has been shown to be a potent inhibitor of HBV in humancells (2.2.15 hepatoma cells transfected with HBV genome). Activity inthis in vitro system is said to correlate well with the activity ofcompounds against HBV when administered to chronic HBV carriers.Concentrations of penciclovir of 1 microgram/ml produced a 50% reductionin HBV DNA by 6 day and a 90% reduction by day 9. These effects wereconcentration dependent.

Penciclovir and famciclovir clearly inhibit duck hepatitis B viralreplication in the Peking duck model. Both penciclovir (at oral doses ofpenciclovir of 20 and 100 mg/kg twice a day) and famciclovir (at oraldoses of 5 and 25 mg/kg twice a day) reduced HBV DNA and DNA polymeraseto undetectable levels within two days of the start of treatment, andmaintained this suppression during the 21 days to treatment. Viralreplication remained suppressed for at least two days after dosing hadstopped, after which both HBV DNA and DNA polymerase levels returned topre-treatment levels.

In a completed double-blind, placebo controlled, single centre pilotstudy in patients with chronic hepatitis B, a 10 day course offamciclovir (250 mg or 500 mg tid) resulted in a greater than 90% fallin HBV DNA levels in 6 of 11 evaluable patients. In an ongoing study inchronic hepatitis B, over 250 patients have received famciclovir at adose of up to 500 mg three times a day for 16 weeks. Preliminary datafrom this study indicate that treatment with famciclovir resulted in asignificant reduction in HBV DNA and ALT levels and was well toleratedwith an adverse events profile similar to placebo. Famciclovir has alsoshown efficacy in a number of named patients with HBV reinfectionfollowing liver transplantation.

There remains a need in the art for improved methods of treatingHepatitis B.

SUMMARY OF THE INVENTION

In accordance with the present invention, treatment of hepatitis B virus(HBV) infection in a patient comprises administering to a patient inneed of such treatment a drug regimen comprising an antiviral-effectiveamount of thymosin α1 (Tα1) and an antiviral-effective amount oflamivudine. In preferred embodiments, the drug regimen comprisesthymosin α−₁, lamivudine and famciclovir.

DESCRIPTION OF THE PREFERRED EMBODIMENTS

A novel modality for treating HBV infection in mammals has been devised,in which a drug regimen is administered to such mammals, the drugregimen including administration of antiviral-effective amounts ofthymosin α−₁ and administration of antiviral-effective amounts oflamivudine. In preferred embodiments, the drug regimen includes thymosinα−₁, lamivudine and antiviral-effective amounts of famciclovir.

Separate dosage units of Tα1, lamivudine and optionally famciclovir, canbe administered to the patient daily, one or more times per day, e.g.,two or three times per day, and doses can be administered one or moredays per week, e.g., two, three, four, five, six or seven days per week.

The terms “thymosin α1” and “Tα1” refer to peptides having the aminoacid sequence disclosed in U.S. Pat. No. 4,079,137, the disclosure ofwhich is incorporated herein by reference.

Antiviral-effective amounts of Tα1 are hepatitis B virus-reducingamounts of thymosin α1 which may be dosage units comprising about 0.5-10mg of thymosin α1. Preferrably, the dosage unit comprises about 1-2 mgof Tα1. Most preferrably, the dosage unit comprises about 1.6 mg of Tα1.

According to one aspect of this embodiment of the present invention, thedosage unit comprising Tα1 is administered to the patient on a routinebasis. For example, the dosage unit can be administered more than oncedaily, once daily, weekly, monthly, etc. The dosage unit may beadministered on a bi-weekly basis, i.e., twice a week, for example, onTuesday and Saturday. Most preferably, the dosage unit of Tα1 isadministered on a thrice weekly basis, i.e., three times per week.

According to another aspect of the invention, the administration of thedosage unit comprising Tα1 is administered for a period of time,concurrent with administration of lamivudine, sufficient to reduce oreliminate HBV infection in the patient.

Lamivudine, as noted above, is 2′3′-dideoxy-3′thiacytidine. According topreferred embodiments, dosage units comprising amounts of lamivudinewhich, in conjunction with administration of Tα1, are effective inreducing hepatitis B virus in a patient, are included within the dosagerange of about 50-500 mg lamivudine. Preferred dosage units comprisinglamivudine include about 100-200 mg lamivudine, most preferably about150 mg lamivudine.

The dosage unit comprising lamivudine can be administered to the patienton a routine basis, for example, the dosage unit can be administeredonce daily, more than once daily (e.g., two, three or more times daily),weekly, monthly, etc. Most preferably, the dosage unit is administeredonce daily.

Administration of the lamivudine dosage unit can occur for a length oftime, in conjunction with administration of Tα1, effective to reduce oreliminate HBV infection in the patient. Preferably, such administrationoccurs until HBeAg of the patient becomes negative, e.g., about 6-12months.

In particularly preferred embodiments, Tol is administered bysubcutaneous injection thrice weekly in pharmaceutical dosage unitswithin the range of about 1-2 mg (e.g., about 1.6 mg). In conjunctionwith the administration to the patient of about 150 mg lamivudineorally, once daily.

According to another aspect of this embodiment, the administration ofthe dosage unit comprising Tα1 is administered for a period of time,concurrent with administration of lamivudine and famciclovir, sufficientto reduce or eliminate HBV infection in the patient.

Famciclovir, as noted above, is the diacetyl ester of6-deoxypenciclovir. According to preferred embodiments, dosage unitscomprising amounts of famciclovir which, in conjunction withadministration of Tα1, are effective in reducing hepatitis B virus in apatient, are included within the dosage range of about 100-2,000 mgfamciclovir. Preferred dosage units comprising famciclovir include about100-1,000 mg famciclovir, most preferably about 250 mg famciclovir.

The dosage unit comprising famciclovir can be administered to thepatient on a routine basis, for example, the dosage unit can beadministered once daily, more than once daily (e.g., two, three or moretimes daily), weekly, monthly, etc. Most preferably, the dosage unit isadministered three times daily.

Administration of the famciclovir dosage unit can occur for a length oftime, in conjunction with administration of thymosin α1, effective toreduce or eliminate HBV infection in the patient. Preferably, suchadministration occurs for at least about six months, and mostpreferably, for about 6-12 months.

In particularly preferred embodiments Tα1 is administered bysubcutaneous injection twice weekly in pharmaceutical dosage unitswithin the range of about 1-2 mg (e.g., about 1.6 mg), in conjunctionwith administration to the patient of about 150 mg lamivudine and 250 mgfamciclovir orally, three times daily.

However, it is to be understood that pharmaceutical dosage unitscontaining Tα1, lamivudine and famciclovir may be formulated in anysuitable manner for administration by any suitable route.

The invention is applicable to native (i.e., naturally occurring Tα1 aswell as synthetic Tα1 and recombinant Tα1 having the amino acid sequenceof native Tα1, amino acid sequences substantially similar thereto, or anabbreviated sequence from thereof, and their biologically active analogshaving substituted, deleted, elongated, replaced, or otherwise modifiedsequences which possess bioactivity substantially similar to that ofTα1.

The invention is applicable to lamivudine, analogs, homologs andderivatives thereof which possess bioactivity substantially similar tolamivudine, biologically active analogs which are capable of conversionto lamivudine or analogs, homologs or derivatives thereof which possessbioactivity substantially similar to lamivudine.

The invention is applicable to famciclovir, analogs, homologs andderivatives thereof which possess bioactivity substantially similar tofamciclovir, biologically active analogs which are capable of conversionto penciclovir or analogs, homologs or derivatives thereof which possessbioactivity substantially similar to penciclovir.

The invention is also applicable to a pharmaceutical combinationeffective in treating hepatitis B virus infection in a patient,including a first dosage unit comprising an antiviral-effective amountof thymosin α1, a second dosage unit comprising an antiviral-effectiveamount of lamivudine and a third dosage unit comprising anantiviral-effective amount of famciclovir.

In preferred embodiments in which both lamivudine and famciclovir areutilized with Tα1 there is a lower probability for the HBV virus tomutate, thereby creating a low viral load period of long duration forthe enhance immunological response of Tα1 to act.

Anti-viral agents such as nucleoside analogs are not viricidal, i.e.,they do not kill viruses. They suppress the replication (reproduction)of the viruses so that the viral load goes down to unmeasurable levels.However, the viruses may not be eliminated altogether by nucleosideanalogs alone.

Viruses, as replicating beings, have the propensity of changing theirgenetic material (DNA in the case of hepatitis B virus) during thereplicative cycle by mutation. Under the pressure of a medication tosuppress their replication, a virus may mutate into a strain which canwithstand the action of the drug, and this tends to occur at a highrate.

If more than one drug is used at the same time, and if the drugs havedifferent modes of action at the molecular level, a virus capable ofsurviving the suppressive action of both drugs would have to mutatesimultaneously at two different points in its DNA. Simultaneous mutationat two different points is much more unlikely than a single mutation.

Thymosin α−₁ is an immune system modulator that can play an instrumentalrole in the activation of host immunity mechanisms for the treatment ofchronic hepatitis B.

Previous studies using Tα1 alone have only been partially satisfactoryin patients where there is initially a very high viral load. Lamivudine,alone or in combination with famciclovir, prevents viral replication,thereby lowering the viral load and enhancing the effect of Tα1.

The invention is further illustrated by the following A examples, whichare not intended to be limiting.

EXAMPLE 1 TREATMENT OF HEPATITIS B INFECTION IN HUMAN PATIENTS

Effacacy of hepatitis B treatment is shown by evaluating the biochemical(ALT), virological (HBV DNA), serological (HbeAg) and histologicalresponse in immune tolerant adult patients with chronic hepatitis Bvirus infection to treatment with Tα1 plus Lamivudine and optionallyFamciclovir.

Efficacy Objectives

The primary endpoints will be the complete virological response ratedefined as the percentage of patients with negative serum HBV DNA (asdetermined by the Chiron Quantiplex™ HBV DNA (bDNA) assay) and HBeAg atthe end of 6-month treatment period and at the end of the 12-monthfollow-up period.

Safety Objectives

This study will evaluate safety data, including clinical status,hematological measures and measures of liver and kidney function, duringthe 6-month treatment period and for 12-month follow-up after the lastadministration of Tα1 plus Lamivudine and optionally Famciclovir.

Study Population

Criteria for Inclusion

1 Age≧18 yrs and ≦65yrs.

2 Either male or female.

3 Documented evidence of the presence of HBsAg in the serum for at leastsix (6) months.

4 ALT<2.5 times the upper limit of normal on 2 determinations 4 weeksapart or the mean of 3 ALTs during the screening phase<2.5 times theupper limit of normal.

5 ALT<100 U/L during the screening phase. 6 HBV DNA>4,000 MEq/ml on 2determinations>4 weeks apart. If the second HBV DNA determination is≦4,000 MEq/ml, a 3rd determination must be done 4 weeks after the 2nd.The 3rd determination must be >4,000 MEq/ml (as determined by the ChironQuantiplex (bDNA) assay).

7 Positive HBeAg on 2 determinations≦4 weeks apart.

8 Liver biopsy within 12 months prior to enrolment consistent withchronic hepatitis.

9 Compensated liver disease with prothrombin time prolonged less than 5sec over control, serum albumin≧30 g/L, bilirubin≦68 mmol/L.

10 Hematocrit≧30%, platelet count≧100×10⁹/L, WBC≦3.5×10⁹/L, andpolymorphonuclear white cell count≦1.7×10⁹/L.

11 Adequate renal function: calculated creatinine clearance≦60 mL/min.

12 If a woman of child-bearing potential, use of an adequate method ofcontraception.

Criteria for Exclusion

1 Concomitant chronic use of any drug known to be hepatotoxic.

2 Concomitant chronic use of any immunosuppressive drug.

3 HIV infection diagnosed by HIV seropositivity and confirmed by Westernblot.

4 Concomitant or prior history of malignancy other than curativelytreated skin cancer or surgically cured in situ carcinoma of the cervix.

5 Active infectious process other than HBV that is not of aself-limiting nature. TB and AIDS are examples of infectious processesthat are not of a self-limiting nature.

6 Cirrhosis.

7 A history of hepatic encephalopathy or bleeding esophageal varices.

8 Pregnancy documented by urine HCG pregnancy test.

9 Intravenous drug and alcohol abuse within the previous 5 years.

10 Patients who are poor medical or psychiatric risks or who have anynon-malignant systemic disease that, in the opinion of the investigator,would make it unlikely that the patient could complete the protocol.

11 Simultaneous participation in another investigational drug study, orparticipation in any clinical trial involving experimental drugs within30 days before study entry.

12 Any indication that the patient would not comply with the conditionsof the study protocol.

13 Previous therapy with interferon or any other type of immunotherapywithin 1 year of entry into the study or treatment with adrenocorticoidsteroids within 6 months of entry into the study.

14 Any other liver disease including hepatitis C, hepatitis delta,alcoholic liver disease, drug-induced liver injury, primary biliarycirrhosis, sclerosing cholangitis, autoimmune hepatitis,hemochromatosis, α1 antitrypsin deficiency, or Wilson's disease.

15 Previous treatment with Tα1.

16 Previous treatment with Famciclovir.

17 Previous treatment with Lamivudine.

18 Patients with known hypersensitivity to Thymosin α1.

19 Patients with known hypersensitivity to Famciclovir.

20 Patients with known hypersensitivity to Lamivudine.

Conduct of Study

Screening Evaluation

All patients will undergo screening evaluation to determine eligibilityfor enrollment into the study. The evaluation consists of two, or onoccasion three, separate screening visits. All data from screeningvisits will be recorded.

First Screening Visit (Screening Visit 1)

A. Eligibility

To be eligible for screening visit 1, the patient must have a history ofchronic hepatitis as evidenced by a history of positive HBsAg for atleast 6 months.

B. Screening Procedures

Laboratory screening tests at screening visit 1 will include HbsAg,HBeAg, hepatitis C antibody, hepatitis Delta antibody, HBV DNA, and ALT.

Complete history and physical examination.

Evaluate liver biopsy obtained within 12 months prior to enrolment.

Second Screening Visit

A. Eligibility

To be eligible to continue to screening visit 2, subjects must havepositive HBsAg, HBeAg, and HBV DNA, and be negative for antibodies tohepatitis C and Delta.

B. Timing

Screening visit 2 will take place no less than 4 weeks after screeningvisit 1, and no more than 2 months after screening visit 1.

C. Screening Procedures

The following tests will be done at screening visit 2:

˜Full blood count (FBC).Includes RBC, hematocrit, hemoglobin, WBC anddifferential counts

˜Platelet count

˜Prothrombin time (PT)

˜Chemistry panel including BUN and creatinine

˜ALT

˜Serum albumin and total protein

˜Bilirubin

˜HbeAg

˜HBV DNA

˜anti-HIV

˜Ferritin

˜Antinuclear antibody

˜α−fetoprotein

˜Urine pregnancy test

Third Screening Visit (Screening Visit 3)

A. Eligibility

A third screening visit will be required only if the value of HBV DNA atscreening 2 is ≧4,000 MEq/ml or if 1 of the ALT values at 1st or 2ndScreening is >2.5 times the upper limit of normal, and the other is <2.5times the upper limit of normal.

B. Timing

Screening visit 3, if required, will take place no less than 4 weeks andno more than 2 months after screening visit 2.

C. Procedures

Laboratory tests at screening visit 3 will include HBV DNA, HBeAg, andALT.

Study Enrolment

Following the screening evaluation, patients will be reviewed todetermine if they meet the inclusion and exclusion criteria.

After informed consent is obtained Patients will be started ontreatment≦4 weeks from the completion of the screening evaluation.

Treatment Phase

Patients will receive treatment with:

Tα1 1.6 mg SQ three times weekly (6 months).

Lamivudine 150 mg QD (once daily, 6 months).

Optionally Famciclovir 250 mg PO TID (three times daily, 6 months).

All subjects will have at least 12-months follow-up observation aftercompletion of therapy.

Specific evaluations to be done during the treatment or observationportions of the study:

Month 0. 1. 3. 6 during treatment then every 6 months for 12 months:

HBV DNA

HBeAg

Anti-HBe(only if HBeAg is negative)

HBsAg

Polyclonal HBsAg (only if HBsAg turned negative by monoclonal test)

Anti-HBc

Anti-HBs (only if HBsAg is negative)

Limited history and limited physical examination

Chemistry panel including: ALT (SGPT), AST (SGOT), alkaline phosphatase,total bilirubin, BUN and creatinine.

Hematology: RBC, hematocrit, WBC, differential, platelet count.

Prothrombin time

Urinalysis (specific gravity, glucose, protein, microscopic)

At month 18:

Repeat liver biopsy

Only at Week 0: urine pregnancy test (postmenarchal female subjectsonly).

Post-treatment Follow-up

Post-treatment follow-up will continue for a minimum of 12 months asspecified above, collecting the data listed.

Definition of Time Limits

When testing every four weeks is required, patients are expected toreturn for scheduled clinic examinations and testing within one week ofthe day specified in the protocol. Missed visits, or visits made morethan one week before or after the scheduled day, will be treated asprotocol violations but these patients will not be excluded from dataanalysis. When testing is scheduled at approximately three-monthintervals, testing should be done within 3 weeks of the specified date.

Study Medication, Supplies, and Packaging

Dosage and Administration

The dose of Tα1 will be standardized at 1.6 mg per injection for alltreated patients who weigh at least 40 Kg. Patients weighing less than40 Kg will receive a dose of 40 ug/kg. This is based on the results ofthe phase II studies in which doses in the range of 1.45 to 1.75 mg (inadults) per twice-weekly injection were found to be safe and effectivein clearing HBV DNA from the blood.

Lamivudine will be given at a dose of 150 mg once daily for allpatients.

FAMVIR (Famicilovir) will optionally be given at a dose of 250 mg threetimes daily for six months.

Dosage Adjustments

No dosage adjustments are planned in this study.

Drug Supplies and Packaging

Synthetic Tα1, which has been formulated with mannitol and sodiumphosphate, is manufactured by or for SciClone Pharmaceuticals insingle-dose vials for injection. Vials will require reconstitution withsterile water for injection. The vials will be labelled with the drugname and dosage. This will be an open study.

Lamuvidine will be provided in 150 mg doses.

FAMVIR will be provided as 250 mg tablets.

All drug supplies must be kept in a secure area, and dispensed only bypharmacists or other research members designated by investigators whohave been approved for participation in this study.

Concomitant Medications and Lifestyle Immunomodulatory drugs (except forthe use of Tα1), glucocorticoids (such as prednisone), immunosuppressivedrugs and drugs known to be hepatotoxic are prohibited.

No restrictions on other concomitant medications or lifestyle will beplaced on the Patient; however, Patients will be discouraged fromexcessive use of alcoholic beverages.

Assessment of Compliance

Compliance with study medication dosing is defined as the Patientreceiving ≧80% of the scheduled amount or study medication each month.

Patients will return to the clinic for each injection of Tα1, andadministration of the dose will be documented by the personadministering it. At the discretion of the investigator, a patient whois likely to be highly compliant with the protocol may make arrangementsfor home, or self administration.

In cases of home administration of drug the patient and/or the patient'sparent(s) and, if so desired, a designated health care worker willreceive instructions on the process of self-injection or assistedinjection by the study nurse. The study nurse will continue toadminister the investigational drug until assured of the patient'sability to self-administer, or of the ability of a parent of designatedassistant to provide the injections. The study nurse will communicatewith the patient or parent each week and record compliance with theinjections. The patient or parent will maintain a diary of theinjections actually given, and of any adverse experiences.

Patients having home injections will be given appropriate container fordisposal of used needles and syringes, and instructed in proper disposaltechniques.

Discharging Patients from the Study

Criteria for Discharging Patients

1. Any treated patient who has an adverse reaction to treatment thatthreatens his/her well being will have treatment discontinued. Thepatient will be monitored for resolution of the adverse event and willcontinue to be monitored on the protocol schedule until completing thestudy.

2. Any patient who demonstrates a significant deterioration in his/herclinical status, in hematological parameters, or in biochemical tests ofliver and/or renal function will be evaluated by the investigator andthe monitoring committee. Evidence that would suggest such adeterioration includes:

a) Progressive increases in ALT or AST over an interval of 6 months.Note that transient elevations in ALT and AST may precede atreatment-related or spontaneous remission, and are not a reason fordischarging the patient from the study.

b) Progressive increases in the total serum bilirubin levels over aninterval of 6 months.

c) Subjective increase in symptomatology so as to preclude the samelevel of daily activity as exercised by the patient at the time ofinclusion.

d) Hematologic and renal parameters outside the ranges listed in theinclusion criteria.

3. Any patient who withdraws voluntarily from the study.

4. Failure of patient, for whatever reason, to comply with studymedication dosing defined as the Patient receiving <80% of the scheduledamount or study medication each month or failure to comply with otherrequirements of the protocol.

5. Withdrawal from the treatment is considered by the investigator to bein the patient's best interest.

6. The patient dies during the study.

7. The patient has completed entire combined 6-month treatment and12-month follow-up period.

Procedure for Handling Dropouts

Patients removed from this study because of noncompliance with studymedication dosing, defined as the Patient receiving <80% of thescheduled amount or study medication each month, will be replaced.

All Patients removed from the study will continue to be followed, andtheir clinical course included in the final report.

Adverse Experiences

Documenting Adverse Experiences

Adverse event information will be documented during the entire combined6-month treatment and 12-month follow-up period. Any adverse eventscontinuing at the time of the last scheduled visit will be followeduntil they are resolved or explained or until the event stabilizes andthe overall clinical outcome has been ascertained.

Patients will be monitored for significant side-effects or allergicmanifestations possibly resulting from treatment. Although no local orsystemic side effects have been observed with Tα1, the injections willbe terminated if systemic hypersensitivity reactions such as urticariaor wheezing occur. Patients will be educated on the symptoms of severeanaphylactic reactions and informed of appropriate countermeasures.

All patients will be requested to report on any problems emerging sincethe previous visit. To avoid observer bias, all patients will be askedby non-directed questions about adverse events throughout the study.Non-directed questions include “Have you had any problems since yourlast visit?” When problems are described, they will be pursued ingreater detail. The investigator will determine if the adverse event canreasonably be related to the study medication. All adverse events willbe recorded, including date of onset, duration, and severity.

Assessment of Severity of Adverse Experiences

The severity of adverse events will be designated as mild, moderate, orsevere as follows:

Mild No clinical significance, no requirement for additional assessmentModerate Event presented a problem, but did not affect daily activitiesor clinical status Severe Event resulted in marked alteration of dailyactivities or clinical status

In addition to classifying the adverse event as mild, moderate, orsevere the Investigator should determine whether or not an event isserious. The regulatory definition of a serious event includes thosethat are fatal, life-threatening (e.g., anaphylaxis), severely orprematurely disabling or incapacitating, or events resulting in orprolonging inpatient hospitalization, congenital anomaly, cancer, or adrug overdose (whether accidental or intentional).

Assessment of Causality

Every effort should be made by the investigator to explain each adverseexperience and assess its relationship, if any, to study drug treatment.Causality should be assessed using the following categories: unrelated,probably related, possibly related, related.

The degree of certainty with which an adverse experiences is attributedto drug treatment (or alternative causes, e.g., natural history of theunderlying diseases, concomitant therapy, etc) will be determined by howwell the experience can be understood in terms of one or more of thefollowing:

1. Known pharmacology of the drug.

2. Reaction of similar nature being previously observed with this drugor class of drug.

3. The experience having often been reported in literature for similardrugs as drug related e.g. skin rashes, blood dyscrasia.

4. The experience being related by time to drug ingestion terminatingwith drug withdrawal (dechallenge) or reproduced on rechallenge.

Follow-Up of Adverse Experiences

Investigators should follow-up subjects with adverse experiences untilthe event has subsided (disappeared) or until the condition hasstabilized. Reports relative to the subject's subsequent course must besubmitted to the clinical study monitor.

Overdose

Any instance of overdose (suspected or confirmed) must be communicatedto the investigator within 24 hours and be fully documented as a seriousadverse experience. Details of any signs or symptoms and theirmanagement should be recorded including details of any antidote(s)administered.

Pregnancy

Subjects who become pregnant during the study should discontinuetreatment immediately.

Subjects should be instructed to notify the investigator if it isdetermined after completion of the study that they become pregnanteither during treatment or within 30 days after the end of treatment.

Whenever possible a pregnancy should be followed to term, any prematureterminations reported, and the status of the mother and child should bereported after delivery.

Administrative Requirements

Review and Consent Requirements

Ethical Review Committee

The sponsor will supply all necessary data to the investigator forsubmission to the Ethics Committee (Institutional Review Board) at theinvestigator's institution.

Ethics and Informed Consent

All patients will sign informed consent forms approved by the hospitalInstitution Review Board. The form will state the nature of the researchstudy, the type of treatment options, the nature of samples to beobtained, and the possible risks and benefits. The investigator or hisdesignee will obtain informed consent after ascertaining that thepatient fully understand the contents of the consent form. A copy of thesigned consent form will be given to the patient. Patientconfidentiality will be maintained throughout the study, and patientswill be identified on case report forms only by assigned studyidentification numbers.

Procedures and Possible Risks

Venipuncture and Phlebotomy

Patients will have approximately 30 ml of blood drawn prior to entryinto the study, approximately monthly initially and every three monthsfor the remainder of the study. For smaller patients, efforts will bemade to draw only the minimum amount of blood required for the testslisted in this protocol. The blood loss resulting from this testing isnot felt to be significant in the patients who will meet the criteriafor inclusion in this study. Risk of venipuncture and phlebotomy will beminimized by use of an experienced person to carry out these procedures,and further minimized by use of aseptic technique.

Liver biopsy

Patients would have percutaneous liver biopsy performed before the entryinto the study and at the end of the 12 months follow-up period. Liverbiopsy would be performed by experienced hepatologist. The incidence ofcomplications is less than 5% and this includes pain at the site ofentry, hemorrhage, bile peritonitis, pneumothorax, penetration ofabdominal viscera and sepsis. The mortality rate is less than 0.1%.

Laboratory

Laboratory studies will be conducted by a certified laboratory of theinvestigator's choosing. Investigators should employ the same laboratoryduring the entire study. The investigator will supply the sponsor with acopy of the laboratory's current certification, a list of the testmethods used, and a list of normal ranges for the tests included in theprotocol. When appropriate, normal values should be listed on age andsex. These must be provided at the onset of the study, and will be usedto interpret results obtained in the study. If it is necessary to changelaboratories during the study, or if the laboratory changes methodologyor normal values, patient records must have the data of these changesnoted. When possible, laboratory methods should not be changed duringthe course of the study.

For certain tests the sponsor may wish to specify a particular testinglaboratory. For instance, determination of HBV DNA may be such a test.This will be negotiated with the investigator.

Data Evaluation

Criteria for Efficacy

Primary Endpoints

The primary endpoints will be the complete virological response ratedefined as the percentage of the patients with negative HBV DNA (asdetermined by the Chiron Quantiplex™ HBV DNA (bDNA) assay) and HBeAg atthe end of the 6-month treatment period and at the end of the 12-monthfollow-up period.

Secondary Endpoints

1. The percentage change from baseline in the levels of HBV DNA at theend of the 6-month treatment and 12-month follow up period;

2. The proportion of patients who have a reduction in their ALT levelsto below the upper limit of the normal range at the end of treatmentperiod and at the end of the 12 month follow up period;

3. The proportion of patients with loss of hepatitis B s antigen at theend of the treatment period and at the end of the 12 month follow upperiod;

4. The proportion of patients with an improvement in Knodell score ofliver histology.

Safety Evaluation

The clinical assessments and frequent blood testing will provide amechanism to monitor patients for drug safety and to minimize the riskof undiscovered adverse reactions.

Statistical Assessment

Analysis

Data will be analyzed by the investigators, and also by SciClonePharmaceuticals, or by its statistical consultants. Data will be testedfor normality, skewness, and heterogeneity of variances. If needed, datawill be transformed using logarithmic function.

Analyses will include:

description and analyses of such demographic variables as age and sex

baseline characteristics such as medical history and physical exam

All significance testing will be done using two-tailed tests, andstatistical significance will be based upon an alpha level of 0.05. Datalistings, cross tabulations, and graphics will be used appropriately tosupport the analyses and the narrative report.

Safety Analysis

Safety assessment will be based upon analysis of observed clinical,local, or systemic effects. The incidence of abnormalities of eachlaboratory results will be presented. Laboratory abnormalities ofindividual patients will be reviewed by the medical monitor according tospecified criteria.

Subgroup Analysis

The following subgroups will be identified, and their results analyzed.The results may not, depend upon the number of patients in each group,be statistically significant. In such instance, the information may beused as a guide to future studies:

1. Male patients; female patients

2. Liver biopsy

patients showing minimal changes

patients showing chronic persistent hepatitis

patients showing chronic active hepatitis

3. Known duration of the patient's carrier state.

4. Patient age.

Example 2

Three adult patients (ages: 35-65) with chronic hepatitis B who arepositive for HbeAg, HbeAg and HBV-DNA (50-250 pg/ml) and elevated ALT toat least twice normal (80-150 μ/l) were recruited to the study.

Treatment scheme: Tα1 1.6 mg thrice weekly for 6 months, plus lamivudine150 mg once daily. The oral medication was continued until HBeAg becamenegative.

Results: All patients had normalization of ALT within three months ofinitiation of treatment. One year after iniation of therapy, allpatients had normal ALT and were negative for HBV-DNA (hybrid capture).Two patients became negative for HBeAg while one remained HBeAgpositive. All patients tolerated the treatment well and there were noserious side effects reported.

Example 3

The aim of this study was to investigate the efficacy of the combinationof Tα1, which increases host immunity and lamivudine and famciclovir,which suppress viral replication.

Eleven patients with confirmed chronic hepatitis B and positive forHBsAg, HBeAg, and HBV DNA with an elevated ALT of at least two timesnormal were entered into the study. Tα1, 1.6 mg was administered SCthrice weekly for six months. Lamivudine, 150 mg QD and famciclovir 250mg TID were administered until the patients became HBeAg negative.

Within one year of total treatment 64% (7/11) of the patientsexperienced a seroconversion of HBeAg to negative with all patientshaving the ALT normalize (11/11). The HBeAg seroconversion ranged intime from four to 11 months. The HBV DNA became negative in allpatients. One patient experienced an ALT elevation of 150 U/L withcontinuation of therapy. However, this patient demonstrated a HBV DNArelapse. All of the patients tolerated the treatment and there were noserious side effects reported.

The combination of Tα1, lamivudine and famciclovir appears safe andeffective in the treatment of chronic hepatitis B.

Positive results of the above study show effacacy of treatment ofhepatitis B virus infection in patients with Tα1, lamivudine andoptionally famciclovir.

Since many modifications, variations and changes in detail may be madeto the described embodiment, it is intended that all matter in theforegoing description be interpreted as illustrative and not in alimiting sense.

What is claimed is:
 1. A method of treating hepatitis B virus (HBV)infection in a patient, comprising administering to a patient in need ofsuch treatment a drug regimen comprising an antiviral-effective amountof Thymosin alpha 1 (Tα1) and an antiviral-effective amount oflamivudine.
 2. The method of claim 1 further comprising co-administeringto said patient an anti-viral effective amount of famciclovir.
 3. Themethod of claim 2 wherein said Tα1 is administered by injection in adosage amount within the range of about 0.5-10 mg, said lamivudine isadministered orally in a dosage amount within the range of about 50-500mg and said famciclovir is administered orally in a dosage amount withinthe range of about 100-2,000 mg.
 4. The method of claim 2 wherein saidTα1 is administered in a dosage amount within the range of about 1-2 mg,said lamivudine is administered orally in a dosage amount within therange of about 100-200 mg and said famciclovir is administered in adosage amount within the range of about 250-1,000 mg.
 5. The method ofclaim 4 wherein said Tα1 is administered to said patient thrice weekly,said lamivudine is administered to said patient once daily and saidfamciclovir is administered to said patient three times daily.
 6. Themethod of claim 4 wherein said Tα1 is administered in a dosage amount ofabout 1.6 mg, said lamivudine is administered in a dosage amount ofabout 150 mg and said famciclovir is administered in a dosage amount ofabout 250 mg.
 7. The method of claim 6 wherein said Tα1 is administeredto said patient thrice weekly, said lamivudine is administered to saidpatient once daily and said famciclovir is administered to said patientthree times daily.
 8. A pharmaceutical combination for use in treatinghepatitis B virus (HBV) infection in a patient, including a first dosageunit comprising an antiviral-effective amount of thymosin alpha 1 (Tα1)and a second dosage unit comprising an antiviral-effective amount oflamivudine.
 9. The combination of claim 8 further comprising a thirddosage unit comprising an antiviral-effective amount of famciclovir. 10.The combination of claim 9 wherein said dosage unit of Tα1 is a dosageamount within the range of about 0.5-10 mg, said dosage unit oflamivudine is a dosage amount within the range of about 50-500 mg andsaid dosage unit of famciclovir is a dosage amount within the range ofabout 100-2,000 mg.
 11. The combination of claim 9 wherein said dosageunit of Tα1 is a dosage amount within the range of about 1-2 mg, saiddosage unit of lamivudine is a dosage amount within the range of about100-200 mg and said dosage unit of famciclovir is a dosage amount withinthe range of about 100-1,000 mg.
 12. The combination of claim 9 whereinsaid dosage unit of Tα1 is a dosage amount of about 1.6 mg, said dosageunit of lamivudine is a dosage amount of about 150 mg and said dosageunit of famciclovir is a dosage amount of about 250 mg.
 13. The methodof claim 1 wherein said Tα−₁ is administered by injection in a dosageamount within the range of about 0.5-10 mg, and said lamivudine isadministered orally in a dosage amount within the range of about 50-500mg.
 14. The method of claim 13 wherein said Tα−₁ is administered in adosage amount of about 1.6 mg, and said lamivudine is administered in adosage amount of about 150 mg.
 15. The method of claim 14 wherein saidTα−₁ is administered to said patient twice weekly, and said lamivudineis administered to said patient once daily.
 16. The combination of claim8, wherein said dosage unit of Tα−₁ is a dosage amount within the rangeof about 0.5-10 mg, and said dosage unit of lamivudine is a dosageamount within the range of about 50-500 mg.
 17. The combination of claim8, wherein said dosage unit of Tα−₁ is a dosage amount of about 1.6 mg,and said dosage unit of lamivudine is a dosage unit of about 150 mg.